Pharmaceuticals for the treatment of rejection reactions in organ transplantations

ABSTRACT

The use of compound 1 and/or 2 of the formulae ##STR1## and of physiologically tolerable salts of compound 2 for the treatment of rejection reactions of the organ recipient to the transplanted organ is described.

This is a division of application Ser. No. 08/997,723, filed Dec. 23,1997, which is a divisional of Ser. No. 07/932,577, filed Aug. 20, 1992now U.S. Pat. No. 5,728,721.

European Patent 13,376 disclosesN-(4-trifluoromethyl)-5-methylisoxazole-4-carboxanilide (compound 1) asbeing anti-inflammatory. Processes for the preparation of this compoundare also described therein.

It is additionally known that the compound 1 and its metaboliteN-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide (compound 2)have immunomodulating properties, so that they are suitable aspharmaceuticals against chronic graft versus host diseases and againstautoimmune disorders, in particular systemic Lupus erythematosus(EP-A-217,206).

U.S. Pat. No. 4,061,767 describes the use of2-hydroxyethyl-idenecyanoacetanilide derivatives for the preparation ofpharmaceuticals having anti-inflammatory and analgesic action.

In the USA, 15,000 organ transplantations were performed in 1990. Themajority of the transplantations relate to the kidney, but hearts, skin,lungs, liver and pancreas are also being increasingly transplanted. In alarge number of the patients, which have been transplanted with an organof another individual a reaction can occure which lead to the rejectionof the grafted organ. A differentiation can be made between three formsof graft rejection:

hyperacute, acute and chronic rejection.

Hyperacute rejection is essentially caused by circulating antibodies inthe blood, which are directed against the tissue of the transplantedorgan (transplant), and in a very short time--often in minutes--lead tonecroses of the transplant.

In acute graft rejection reaction is somewhat delayed. Further, thechronic form of graft rejection can lead to a diseasestate. In this casethe transplants survive the first year after transplantation, but can berejected in the course of the next few years. It is additionally knownthat the transplant-host relationship is not restricted to rejection bythe host organism alone; in certain cases an immune reaction originatingfrom the transplant and directed against the host tissue can occur(EP-A-217,206). A differentiation is therefore made between a rejectionbetween transplant and host and between host and transplant.

It is addition ally known that transplanted organs from different animalspecies for example from the mouse to the rat are also rejected (Roittet al., Immunology, Gower Medical Publishing Ltd., 1985).

To date, no pharmaceuticals are known which offer effective protectionagainst hyperacute rejection reaction. In the clinic, until now donorsand recipients of organs have been tested for incompatibility. In 20 to40% of all patients waiting for a transplant, do not quality for a donororgan. The acute rejection reaction can be treated, but the medicationshow side effects such as nephrotoxicity during treatment. To date,there are also no medicaments know n which can treat the cause of thechronic graft rejection.

The essential pathogenic factor for tissue death in the transplant isregard ed to be allophilic and xenophilic antibodies (Auchincloss H.,Transplantation 46, 1, 1988). These antibodies are essentiallyresponsible for rejection in organ transplants within an animal species(allo) or between two different species (xeno).

Surprisingly, compound 1 and its metabolite, the above-mentionedcompound 2, show a potent inhibition of the formation of allophilic orxenophilic antibodies. There is thus the possibility of effectivelytreating the hyperacute, acute and chronic rejection reaction of therecipient to the transplanted organ.

The invention therefore relates to the use ofN-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxanilide andN-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide and/or theirphysiologically tolerable salts for the preparation of pharmaceuticalsfor the treatment of graft rejection reactions of the organ recipient tothe transplanted organ.

Suitable physiologically tolerable salts of compound 2 are, for example,alkali metal, alkaline earth metal or ammonium salts, including those ofphysiologically tolerable organic ammonium bases. The term organ isunderstood as meaning all organs in mammals, in particular the human,for example kidney, heart, skin, liver, pancreas, muscle, bone,intestine or stomach, but also blood or hair.

Rejection reaction means all defence mechanisms of the recipientorganism which, in the end, lead to cell or tissue death of thetransplanted organ or affect the viability of the transplanted organ.

The compounds 1 and 2 can be prepared by the following process:

A compound of the formula I ##STR2## in which X represents a halogenatom, preferably chlorine or bromine, is reacted with the amine of theformula II ##STR3## to give the compound 1, and this can then be reactedin the presence of a basic agent to give the compound 2.

The abovementioned reactions are carried out under standard conditionsin a known manner (EP-B-13,376, U.S. Pat. No. 4,061,767).

The starting substances for the reactions are known or can be easilyprepared by methods known from the literature.

The invention also relates to pharmaceuticals which contain an effectiveamount of compound 1 or compound 2 and/or physiologically tolerablesalts of compound 2, in addition to pharmaceutically suitable andphysiologically tolerable excipients, diluents and/or other activesubstances and auxiliaries.

The invention also relates to a process for the preparation of apharmaceutical for the treatment of rejection reactions of the organrecipient against the transplanted organ, which comprises bringingcompound 1 or 2 and/or a physiologically tolerable salt of compound 2into a suitable administration form using a pharmaceutically suitableand physiologically acceptable excipient and, if appropriate, othersuitable active substances, additives or auxiliaries.

The pharmaceutical according to the invention can be administeredorally, topically, rectally, intravenously or alternativelyparenterally. Administration is carried out before, during and afterorgan transplantation in the recipient and/or donor.

Suitable solid or liquid pharmaceutical administration forms are, forexample, granules, powders, coated tablets, tablets, (micro)capsules,suppositories, syrups, juices, suspensions, emulsions, drops orinjectable solutions and preparations having a protracted release ofactive substance, in whose preparation customary auxiliaries, such asexcipients, disintegrants, binders, coating agents, swelling agents,lubricants, flavorings, sweeteners or solubilizers are used. Commonlyused auxiliaries which may be mentioned are, for example, magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, cellulose and its derivatives, animal andvegetable oils, polyethylene glycols and solvents, such as, for example,sterile water and mono- or polyhydric alcohols, for example glycerol.

Preferably, the pharmaceutical preparations are prepared andadministered in dosage units, each unit containing as the activeconstituent a certain dose of compound 1 or 2 and/or physiologicallytolerable salts of compound 2. In the case of solid dosage units, suchas tablets, capsules or suppositories, this dose can be up to about 300mg, but preferably 10 to 200 mg.

For the treatment of a patient (70 kg) to whom an organ has beentransplanted, in the early phases after transplantation an intravenousinfusion treatment of at most 1200 mg per day and in the laterrehabilitation phases an oral administration of 3 times 300 mg per dayof compound 1 or 2 and/or of the corresponding salts of compound 2 areindicated.

Under certain circumstances, however, higher or lower doses may also beappropriate. The administration of the dose can be carried out both bysingle administration in the form of an individual dosage unit or elseseveral smaller dosage units and by multiple administration ofsubdivided doses at specific intervals.

Finally, compound 1 or 2 and/or its corresponding salts can also becombined during the preparation of the abovementioned pharmaceuticaladministration forms together with other suitable active substances, forexample antiuricopathics, thrombocyte aggregation inhibitors, analgesicsand steroidal or non-steroidal anti-inflammatories.

EXAMPLE 1

Pharmacological tests and results

2 to 3 month-old rats (LEW) are intraperitoneally (i.p.) sensitized with2×10⁷ human peripheral blood lymphocytes. The i.p. administration ofcompound 2 starts 4 days before sensitization and ends 10 days aftersensitization of the rats. Serum samples are taken from the tail veinand stored at -80° C.; the activity of complement is avoided by heatdeactivation.

The naturally occurring xenophilic antibodies (NXA) and the xenophilicantibodies induced by sensitization (SXA) are titrated and live humanperipheral blood lymphocytes are added (45 min. at 20° C.). Afterintensive washing, FITC-labeled goat-anti-rat IgG or IgM antibodies areadded and the binding to SXA or NXA is quantitatively determined by flowcytometry (FACScan, Becton Dickinson).

A) Non-sensitized rats

The serum of male LEW rats contain NXA which bind to vital humanperipheral blood lymphocytes, to be precise, usually with an average IgMtiter of 1:4 and an average IgG titer of less than 1:1. The rats (n=8)which are treated every day with 10 mg/kg of compound 2, show a 30%reduction in their IgG titer and a 50% reduction in their IgM titer onthe 11th day.

B) Sensitized rats

In sensitized rats there are greatly increased amounts of SXA The IgGtiter is 1:1024 to 1:16384 and the IgM titer 1:4 to 1:256. The IgG titerremains stable for over 50 days, while the IgM titer slowly decreasesafter the 10th day.

The sensitized rats (n=5) are treated with 3 or 10 mg/kg of compound 2.The following SXA titers are tound on the 11th day:

    ______________________________________                                        Compound                                                                        (mg/kg) IgG IgM                                                             ______________________________________                                        0               1:2048  1:90                                                    3 1:25   1:16                                                                 10 1:1    1:1.5                                                             ______________________________________                                    

The action of compound 2 on the formation of allophilic antibodies

Male Brown-Norway rats with a weight of 200 to 250 g are used as heartdonors. The hearts are transplanted into male Lewis rats of the sameweight. All rats are supplied with a standard diet and water and kept inan environment with 12 hours of light and darkness.

On the day of heart transplantation and then at intervals of 4 days, 1ml of blood in each case is taken from the tail vein of the recipientrats (Lewis rats). The titer of allophilic antibodies in the blood ofthe recipient rats is determined by incubating various dilutions of therecipient blood with spleen cells of the donor rats (Brown-Norway rats)and rabbit complement. The cytotoxicity is determined by the Trypan Blueexclusion method. The p value f or the antibodies is determined usingthe standard t-test and compared with the untreated control animals.

Treatment with compound 2 begins on the 4th day after transplantation.10 mg/kg of compound 2 in each case are administered i.p. once per day.The number of animals which are simultaneously treated is 5 (n=5).Treatment ends on the 17th day. The following table shows the mean ofthe cytotoxicity values (%) during the treatment.

    ______________________________________                                                                      p value                                           serum Days after transplantation  on the                                    Dilution                                                                              0       4     7     11  14    17  17th day                            ______________________________________                                        1:5     3       63    36    19  22    11  0.001                                 1:25 3 58 31 16 11 6 0.001                                                    1:125 4 34 20 9 6 5 0.050                                                   ______________________________________                                    

EXAMPLE 2

Preparation of N-(4-trifluoromethyl)-5-methylisoxazole-4-carboxanilide

(Compound 1) ##STR4##

A solution of 0.05 mol of 5-methylisoxazole-4-carbonyl chloride (7.3 g)in 20 ml of acetonitrile is added dropwise at room temperature to asolution of 0.1 mol of 4-trifluoromethylaniline (16.1 g) in 150 ml ofacetonitrile. After stirring for 20 minutes, the precipitated4-trifluoromethylaniline hydrochloride is filtered off with suction andwashed twice with 20 ml of acetonitrile each time, and the combinedfiltrates are concentrated under reduced pressure. 12.8 g of white,crystalline N-(4-trifluoromethyl)-5-methylisoxazole-4-carboxanilide(compound 1) are thus obtained.

EXAMPLE 3

Preparation of N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide(compound 2) ##STR5##

0.1 mol of N-(4-trifluoromethyl)-5-methylisoxazole-4-carboxanilide isdissolved in 100 ml of methanol and treated at +10° C. with a solutionof 0.11 mol (4.4 g) of sodium hydroxide in 100 ml of water. The mixtureis stirred for 30 minutes and, after diluting with water, is acidifiedwith concentrated hydrochloric acid. The precipitated crop of crystalsis filtered off with suction, washed with water and dried in air.

The yield is 24.4 g ofN-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide (compound 2).

Melting point from methanol 205 to 206° C.

EXAMPLE 4

Acute toxicity after intraperitoneal administration

The acute toxicity after intraperitoneal administration of the testsubstances was determined with NMRI mice (20 to 25 g) and SD rats (120to 195 g). The test substance was suspended in a 1% strength sodiumcarboxymethylcellulose solution. The different dosages of the testsubstance were administered to the mice in a volume of 10 ml/kg of bodyweight and to the rats in a volume of 5 ml/kg of body weight. Perdosage, 10 animals were used. After 3 weeks, the acute toxicity wasdetermined by the method of Litchfield and Wilcoxon. The results aresummarized in the table.

                  TABLE                                                           ______________________________________                                                   Compound 1                                                                             Compound 2                                                  acute toxicity acute toxicity                                                 intraperitoneal intraperitoneal                                               LD.sub.50 (mg/kg) LD.sub.50 (mg/kg)                                         ______________________________________                                        NMRI mouse   185 (163-210)                                                                            150 (100-200)                                           SD rat 170 (153-189)                                                        ______________________________________                                    

What is claimed is:
 1. A method comprising the treatment of hyperacuteand chronic rejection reactions of an organ recipient to a transplantedorgan, which comprises administering to said organ recipient in need ofsuch treatment an effective amount of a pharmaceutical compositioncontaining as an active ingredient at least one compound of the formulaI or II: ##STR6## or the compound of formula II in the form of aphysiologically tolerable salt.
 2. The method as claimed in claim 1,wherein said pharmaceutical composition is administered to said organrecipient before transplantation.
 3. The method as claimed in claim 1,wherein said pharmaceutical composition is administered to said organrecipient during transplantation.
 4. The method as claimed in claim 1,wherein said pharmaceutical composition is administered to said organrecipient after transplantation.
 5. The method as claimed in claim 1,wherein said pharmaceutical composition is also administered to theorgan donor.